1. Field of the Invention
This invention relates to the long-term storage of biological materials in a dry state. Particularly, it relates to a method of protecting biological materials from reactions such as oxidation and free-radical attack which can destroy such materials when they are stored for long periods of time, while at the same time protecting them from denaturation and other destruction caused by the drying process itself.
2 Description of the Related Art
Although it is advantageous to be able to store proteins, nucleic acids, cells, and other useful biological products in a dry or frozen state, most such materials lose a significant part of their biochemical activity when subjected to the freezing or drying process. Hence, a body of work has developed in the area of protecting biological materials from the stresses created by the processes of freezing and drying. Most of such work has dealt with freezing and freeze-drying, and only a few studies address the problems involved in air-drying. The patents of Crowe, et al. (U.S. Pat. No. 4,857,316) and Carpenter, et al. (U.S. Pat. Nos. 4,806,343 and 4,897,353) disclose methods of protecting liposomes and proteins, respectively, from the stresses of freezing and freeze-drying. The same group of researchers has also published several papers on the subject, including "Stabilization of Dry Phospholipid Bilayers and Proteins by Sugars" (Biochem. J., vol. 242 (1987), pp. 1-10), "Modes of Stabilization of a Protein by Organic Solutes During Desiccation", Cryobiology, 25:459-470 (1988), and "Cryoprotection of Phosphofructokinase with Sugars During Freeze-Drying . . . ", Biochimica et Biophysica Acta, 923:109-115 (1987). Another group has proposed the use of vitrification as a mechanism to protect blood and whole organs when they are subjected to freezing for long-term storage (Fahy, G. M., "Prospects for Vitrification of Whole Organs", Cryobiology, 18:617-622 (1981); Fahy, G. M., "The Relevance of Cryoprotectant Toxicity to Cryobiology", Cryobiology, 23:1-13 (1986); Fahy, G. M., "Cryopreservation of Biological Materials in a Non-Frozen or Vitreous State," U.S. Pat. No. 4,559,298). (The above publications are hereby incorporated by reference.) However, this proposal has never been extended to other substances, nor to drying, which is not at all feasible for the storage of whole organs.
All of the aforementioned publications focus on the processes of freezing and freeze-drying. The process of drying by exposure to a dessicating material at temperatures above freezing (usually room temperature or above), herein referred to as "air-drying", subjects a biological material to a different set of stresses than those of freezing or freeze-drying. See Crowe, "Are Freezing and Dehydration Similar Stress Vectors? A Comparison of Modes of Interaction of Stabilizing Solutes with Biomolecules", Cryobiology 27:219-231 (1990). This is further demonstrated by the fact that the processes of freeze-drying and air-drying do not have identical effects on biological materials. Whereas sorbitol, a non-vitrifying substance, will protect some biological materials (particularly chloroplast fragments known as thylakoids) from the stresses of freeze-drying, it does not so protect the material during the air-drying process (see experimental results below). Furthermore, whereas the Carpenter patent teaches that transition-metal ions are necessary to protect many proteins when freeze-drying, the work of Roser (U.S. Pat. No. 4,891,319) demonstrates that metal ions are not necessary to protect proteins from the stresses of air-drying.
The aforementioned references, including the Roser patent, address the destructive effects of the preservation process itself, primarily the destruction of the liposome structure and the denaturation of proteins. Prior to the work of the present inventors, little or no work had been done to investigate means for protecting materials from destructive reactions which take place after drying, such as oxidation and free-radical attack. Carpenter discusses the desirability of using a non-reducing sugar as a protectant in his system to avoid the oxidation reaction, but does not propose a method of protecting the proteins from such a reaction when they must be stored in the presence of a reducing sugar.